The Human Immune Monitoring Center (HIMC) proposes to acquire and co-develop one of the world's first mass spectrometry-based systems for single-cell analysis using unique mass tags;this system is known as Cytometry via Time of Flight or CyTOF. With this technology, we will have the capacity to measure up to one hundred parameters per cell. Current state-of-the-art flow cytometers allow us to detect up to 15 parameters per cell. The machine will significantly advance the research goals of many researchers at Stanford University and will enrich the field of immunology as the technology is effectively implemented. With the CyTOF, we have already demonstrated effective staining of 14 markers (four phospho-proteins and 10 cell-surface markers, see Project 1) and we are preparing for an imminent 30-color test. With this device, in a single tube and stain, we will be able to readily detect and characterize all the major cell subsets in the blood (using about 20 different surface molecules), including each and every major stem cell progenitor compartment, and then be able to observe within each of those subsets another 80 intracellular kinase events or other intracellular markers! We will be able to map leukemia patient cell subsets in a complete manner with the goal of informing the clinician of how many tumors the patient has, their state of differentiation, and their response to treatment. Notably, the CyTOF machine does not require compensation, allowing application of statistical techniques;this has been impossible given the constraints of fluorescence noise with traditional machines. The new mass spectrometer-based flow cytometer offers an unparalleled revolution in our divination of immune system processes. The three to five color "traditional" flow cytometer defined the major cell subsets of the immune system we understand today (T cells, B cells, macrophages, etc.). The eight-color machine allowed us to characterize immune system stem cells and led to important advances in stem cell biology and identification of cancer stem cells. By merging this capability with intracellular staining, high-end machines now allow 15-color analysis opening possibilities for patient stratification of drug response outcomes (in AML, JMML, and FL, Nolan &Levy laboratories). Within the year, the CyTOF will enable us to measure 30 simultaneous parameters;in two years we expect to routinely monitor as many as 50 parameters of a single cell (as many as 100 might be accomplished with an additional chelator chemistry enables to bind additional ions). This will allow us to dissect biological mechanisms of normal human development and those of many disease processes that are impacted by the immune system. Finally, as this technology is developed it will allow us to hire additional technical staff to develop and run the machines, thus creating a new class of jobs for the HIMC as well as, very soon after that, the Stanford Shared FACS Facility. PUBLIC HEALTH RELEVANCE: We will develop an advanced, hybrid mass spectrometer/flow cytometer that vastly increases the number and precision of measurements one can achieve in flow cytometric applications. We will use this tool to enhance our understanding of immune mechanisms in cancer, infection, and immunity. This instrumentation will likely revolutionize biomedical applications to an extent similar or greater than what traditional flow cytometry has previously achieved.